Bacterial restriction-modification (R-M) systems are diverse in specificity and strategy, but their general function is to protect bacteria from foreign DNA, such as DNA from bacteriophages. R-M systems can consist of a DNA methyltransferase and a restriction endonuclease. DNA methyltransferases catalyze the transfer of a methyl group from the donor S-adenosyl-L-methionine (also known as “SAM” or “AdoMet”) onto adenine or cytosine residues within particular DNA sequences of the host bacterium, which are called recognition sequences. There are three major classes of DNA methyltransferases, classified according to the nature of the product they produce. The first class consists of amino-methyltransferases which catalyze the methylation of the exocyclic amino group of adenine to form the product N6-methyladenine. The second class consists of amino-methyltransferases that catalyze the formation of the exocyclic amino group of cytosine to form the product N4-methylcytosine, while the third class consists of methyltransferases that methylate the cyclic carbon-5 atom of cytosine to form 5-methylcytosine. These methylated bases serve important functions in bacterial R-M systems, as they protect the host chromosome against the otherwise deleterious action of the partner restriction enzyme, which cleaves unmethylated recognition sequence DNA but ignores fully methylated DNA. Thus, it is the combined action of the DNA methyltransferase and its cognate restriction endonuclease that protects the host bacterium from any unmodified foreign DNA. While R-M systems perform an important protective function, they also inhibit the transfer of plasmids between bacterial species and even between strains of the same species of bacteria, as multiple R-M systems within a single bacterial strain can all participate in the restriction barrier. Thus, R-M systems act as a barrier for the genetic manipulation of many bacteria, including the biotechnologically important genus Clostridium. 
The genus Clostridium consists of a large number of species with a wide range of biochemical and physiological traits. See Cato et al., 1986, Genus Clostridium, pp. 1141-1200, in P. H. Sneath et al. (eds.), Bergey's Manual of Systematic Bacteriology, Vol. 2, Williams and Wilkins, Baltimore, Md. There are four criteria that need to be met for an isolate to be assigned to the genus Clostridium: (1) the ability to form endospores, (2) anaerobic energy metabolism, (3) the inability for dissimilatory sulfate reduction, and (4) possession of a Gram positive cell wall. See Andresson et al., 1989, Introduction to the physiology and biochemistry of the genus Clostridium, pp. 27-62, in Minton and Clarke (eds.), Clostridia, Plenum Press, New York. Acetogenic bacteria of the genus Clostridium use synthesis gas (syngas) as a source of carbon and reducing power for growth under anaerobic conditions. Syngas is composed of a mixture of H2, CO and CO2, which is produced by gasification of any organic material, from municipal waste to agricultural by-products. The use of syngas as a feedstock for the biological production of commodity enzymes and chemicals is attractive due to its low cost and the breadth and flexibility of sources from which it is derived. However, the acetogens within the genus Clostridium are relatively uncharacterized, and the ability to genetically manipulate these organisms, particularly through the introduction of heterologous nucleic acids that are stable and not cleaved by clostridial restriction endonucleases, is largely undeveloped. The ability to transform clostridial bacteria is a necessary and fundamental first step for their effective use in the production of industrial bio-products (e.g, isoprene, butadiene and ethanol).
Efforts to overcome R-M systems in Clostridium have typically involved the in vivo methylation of heterologous DNA prior to its transformation to protect it from degradation by restriction endonucleases in the host cells; for example, methylation can be performed in vivo by transforming shuttle plasmids into a strain (e.g., E. coli) expressing one or more heterologous methyltransferases (e.g., a methyltransferase from Bacillus subtilis phage Φ3T). After the methylated DNA is isolated, it may be transformed into host anaerobic cells (e.g, Clostridium aceticum cells) via electroporation, protoplast transformation, conjugal transformation, gene gun, or other method known in the art.
Other methods of overcoming clostridial R-M systems involve the methylation of heterologous DNA in vitro using one or more purified methyltransferase enzymes available for purchase from commercial vendors (e.g., New England BioLabs), or involve the creation and use of clostridial host cells deficient in at least one restriction endonuclease gene in their restriction-modification system. See, e.g., Dong et al., PLoS ONE 2010 5(2):e9038. In Dong et al. (2010), a putative type II restriction endonuclease (Cac824I), identified from the publicly-available genome of Clostridium acetobutylicum ATCC 824, was disrupted using the ClosTron group II intron insertion-based gene knockout system. The ClosTron system, similar to most group II intron approaches, uses an element derived from the broad host range LI.LtrB intron of Lactococcus lactis. See, e.g., Kuehne et al., 2011, ClosTron-mediated engineering of Clostridium. Methods in Molecular Biology, Vol. 765:389-407. The resulting cells deficient in Cac824I could be transformed with unmethylated DNA (e.g., unmethylated plasmid DNA) via electroporation.
However, these processes for overcoming the restriction-modification systems in clostridial bacteria depend upon the identification of the specific methyltransferases and restriction endonucleases present in the clostridial bacteria of interest. For example, in order to transform a clostridial bacterial species with a plasmid of interest, treating the desired plasmid in vivo or in vitro with a heterologous methyltransferase (e.g., with Bacillus subtilis phage Φ3T methyltransferase) will only protect the plasmid from cleavage if the restriction endonuclease inside the host cell has the same DNA recognition sequence as the heterologous methyltransferase. To improve the effectiveness of such an approach, multiple heterologous methyltransferases, each with different DNA recognition sequences, may be used; however, this increases the time and cost of each attempted transformation. If the methyltransferases used do not recognize the same sequence as the restriction endonuclease present inside the clostridial cell of interest, the heterologous DNA will not be protected from cleavage.
Accordingly, there remains a need to identify and circumvent restriction-modification systems in clostridial bacteria to facilitate their use in the production of industrial bio-products including, but not limited to, isoprene, butadiene, and ethanol.
Throughout the specification, various publications (including sequences), patents, and patent applications are disclosed. All of these are hereby incorporated by reference in their entirety for all purposes.